This invention relates to methods of inducing disease resistance in plants.
Plant-Pathogen Interactions
When plants are attacked by pathogens, they mount a battery of defenses, including senescence of infected tissues, reinforcement of cell walls by crosslinking and synthesis of new cell wall components, and production of antimicrobial compounds, such as phytoalexins, defensins, and enzymes that degrade pathogen cell walls (reviewed in Gan et al., Plant Physiology 113:313–319, 1997; Glazebrook, Current Opinion in Plant Biology 2:280–286, 1999; and Somssich et al., Trends in Plant Science 3:86–90, 1998). If the plant and pathogen express resistance (R) and avirulence (avr) genes, respectively, which interact to trigger a hypersensitive response, the plant is able to successfully resist the pathogen and the relationship is termed “incompatible.” In contrast, in a “compatible” interaction, the plant fails to resist the pathogen attack and disease ensues. Although many of the same defenses that are induced during an incompatible interaction are also activated during a compatible interaction, the induction is generally slower and/or less extensive (reviewed in Crute et al., Arabidopsis, Cold Spring Harbor Laboratory Press pp. 705–747, 1994; Draper, Trends in Plant Science 2:162–165, 1997; Van Camp et al., Trends in Plant Science 3:330–334, 1998; and Yang et al., Genes & Development 11:1621–1639, 1997), and the plant's defense response is insufficient to prevent colonization by the pathogen. However, in many compatible interactions, the plant is nonetheless able to limit pathogen propagation, as was demonstrated by the isolation of Arabidopsis mutants that are defective in defense-related processes, and consequently allow enhanced growth of virulent pathogen(s) (Glazebrook et al., Genetics 143:973–982, 1996; Rogers et al., The Plant Cell 9:305–316, 1997; and Volko et al., Genetics 149:537–548, 1998).
Role of Salicylic Acid in Plant Defense Response
Salicylic acid (SA) is a crucial signaling molecule in the plant defense response to pathogen attack (Enyedi, et al., Cell 70:879–886, 1992). For example, SA plays an important role in so-called systemic acquired resistance, or SAR (Gaffney et al., Science 261:754–756, 1993). SAR is a phenomenon in which infection of a plant with a pathogen that activates R gene-mediated pathways leads to accumulation of PR proteins, such as PR1, BGL2, and PR5 in uninfected leaves, which concomitantly become resistant to a variety of pathogens (Enyedi, et al., supra and Malamy et al., Plant J. 2:643–654, 1992). Treatment of plants with SA leads to PR protein accumulation and pathogen resistance (Enyedi et al. supra and Malamy et al., supra). Importantly, localized SA-mediated activation of PR proteins is also involved in defense responses to virulent pathogens that do not elicit a localized plant defense response, known as the hypersensitive response (HR), illustrating how R gene-dependent and R gene-independent pathways can utilize some of the same signaling compounds and effectors (Reuber et al., Plant J. 16:473–485, 1998 and Zhou et al., Plant Cell 10:1021–1030, 1998). The role of SA signaling in SAR and other plant defense responses has been facilitated by the construction of transgenic plants that express a Pseudomonas putida gene, nahG, which depletes the endogenous pool of SA by converting it into catechol (Gaffney et al., supra). These so-called “nahG transgenic plants” fail to exhibit SAR and are more susceptible to a variety of bacterial and fungal pathogens (for example, see Gaffney et al., supra and Reuber et al., supra). Several Arabidopsis genes have been identified that affect SA signaling pathways. For example, mutant alleles of NPR1, which encodes a protein with ankyrin repeats (Cao et al., Cell 88:57–64, 1997 and Ryals et al., Plant Cell 9:425–439, 1997), fail to activate SAR and PR gene expression in response to SA and exhibit enhanced susceptibility to pathogens (Cao et al., Plant Cell 6:1583–1592, 1994; Delaney et al., Proc. Natl. Acad. Sci. U.S.A. 92:6602–6606, 1995; Glazebrook, et al., 1996; and Shah et al., Mol. Plant-Microbe Interact. 10:69–78, 1997). Two additional Arabidopsis genes that are involved in SA signaling are PAD4 and EDS5. pad4 and eds5 plants were identified on the basis that they are more susceptible to virulent pathogens such as Pseudomonas syringae and Erysiphe orontii (Glazebrook et al., 1996) and accumulate decreased levels of SA following pathogen attack (Zhou et al., supra and Nawrath et al., Plant Cell 11:1393–1404, 1999). eds5 is allelic to an independently isolated mutant called sid1, isolated on the basis of reduced SA accumulation in response to pathogen attack (Nawrath et al., supra). Studies using eds5/npr1 double mutants suggest that EDS5 operates in a SA-dependent, but NPR1-independent, pathway (Reuber et al., supra).
In addition to SA, recent studies have identified SA-independent resistance mechanisms in Arabidopsis that are mediated by jasmonic acid (JA) and ethylene (ET) (reviewed in Dong, Curr. Op. Plant Biol. 1:316–323, 1998 and Reymond et al., Curr. Op. Plant Biol. 1:404–411, 1998). For example, JA induces the accumulation of the antimicrobial peptides thionin and defensin, encoded by THI and PDF genes, respectively. This induction is blocked in the ethylene insensitive mutant ein2 and in the jasmonate-insensitive mutants jar1 and coi1, but is not affected in nahG transgenic plants (Penninckx et al., Plant Cell 8:2309–2323, 1996; Penninckx et al., Plant Cell 10:2103–2113, 1998; and Thomma et al., Proc. Natl. Acad. Sci. U.S.A. 95:15107–15111, 1998). The JA pathway, rather than SA-mediated pathways, appears to be particularly important in conferring resistance to necrotrophic fungal pathogens (Penninckx et al., 1996 and Thomma et al., 1998). The SA and JA pathways appear to be at least partially antagonistic (Dong, supra and Pieterse et al., Plant Cell 10:1571–1580, 1998). Another SA-independent but JA- and ET-dependent pathway is called ISR for induced systemic resistance (Pieterse et al., Plant Cell 8:1225–1237, 1996 and Pieterse et al., 1998). Interestingly, ISR, which is triggered by the biocontrol bacterium Pseudomonas fluorescens in association with Arabidopsis roots, is dependent on NPR1, indicating that the JA/ET and SA resistance pathways intersect (Pieterse et al., Trends Plant Sci. 4:52–58, 1999 and Pieterse et al., 1998).
Biosynthesis of Salicylic Acid
Early studies showed that salicylic acid in higher plants derives from the shikimate pathway (Zenk et al., Planzen. Z. Naturforsch 19B:398–405, 1964). The shikimate pathway occurs in microorganisms and plants, but not in animals (reviewed in Herrmann et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 50:473–503, 1999). The shikimate pathway links the metabolism of carbohydrates to the biosynthesis of aromatic compounds. Chorismate, the end product of this pathway, is the precursor of the aromatic amino acids, tryptophan, phenylalanine, and tyrosine, and a diverse array of aromatic secondary compounds. In higher plants, the shikimate pathway appears to be localized to the plastid, because shikimate pathway cDNAs encode proteins with amino terminal plastid import sequences (Id.). In addition, the chorismate-utilizing enzymes, anthranilate synthase (AS), chorismate mutase (CM), and isochorismate synthase (ICS) also contain putative plastid import sequences (Eberhard et al., FEBS Lett. 334:233–236, 1993; van Tegelen et al., Plant Physiol. 119:705–712, 1999; and Zhao et al., J. Biol. Chem. 270:6081–6087, 1995). The biosynthesis of salicylic acid in tobacco, cucumber, and potato has been deduced to occur via the conversion of phenylalanine to benzoic acid (Pathway 1 in FIG. 1). Evidence for this biosynthetic pathway includes (1) the dependence of SA biosynthesis on phenylalanine ammonia lyase (PAL) activity, (2) numerous radiolabeling studies, and (3) the isolation of a TMV-inducible benzoic acid 2-hydroxylase activity (BA2H) in tobacco (Coquez et al., Plant Physiol. 117:1095–1101, 1998; Leon et al., Plant Physiol. 103:323–328, 1993; Meuwly et al., Plant Physiol. 109:1107–1114, 1995; Ribnicky et al., Plant Physiol. 118:565–572, 1998; and Yalpani et al., Phytopathology 83:702–708, 1993). Though BA2H activity was first identified in 1993 (Leon et al., supra), the cloning of the corresponding gene has not been reported.
Pseudomonas aeruginosa synthesizes salicylic acid from chorismate utilizing isochorismate synthase (PchA) and pyruvate lyase (PchB) genes (Serino et al., Mol. Gen. Genet. 249:217–228, 1995). It should be noted, however, that many bacteria contain isochorismate synthases, and that these enzymes are involved in processes other than salicylic acid production. For example, in E. coli, there is an isochorismate synthase required for menaquinone production (MenF), and another involved in enterobactin (a siderophore (i.e., an iron-binding compound)) synthesis (EntC). In the case of Pseudomonas aeruginosa, PchA is the first enzyme in the pathway leading to production of the siderophores salicylic acid and pyochelin. An elicitor-inducible isochorismate synthase was isolated from plants and the sequence of cDNA clones from C. roseus and A. thaliana were reported (van Tegelen et al., supra and Meng et al., Plant Physiol. 118:1536, 1999). In addition, two putative isochorismate synthases were annotated in the Arabidopsis thaliana genome (Accession No. AAF15941 (referred to as “At ICS1”; SEQ ID NO.: 1) and Accession No. AAF27094 (referred to as “At ICS2”; SEQ ID NO.:2). However, as is described in greater detail below, this patent provides the first proof in plants that an alternate salicylic biosynthetic pathway exists and that this alternate pathway 1) requires isochorismate synthase (specifically the inducible At ICS1) and 2) is involved in plant defense against pathogens.
Powdery Mildews as Pathogens
Powdery mildews infect numerous plant species and cause extensive crop loss (Agrios, Plant Pathology, Academic Press, San Diego, 1997). As obligate biotrophic fungal pathogens, they require specific host signals for development. Consequently, they are useful organisms for studying both host factors that facilitate disease development and host defense responses that limit disease. Three species of powdery mildew that infect Arabidopsis have been identified: Erysiphe cichoracearum (Adam et al., The Plant Journal 9:341–356, 1996), E. cruciferarum (Koch et al., Bot. Helv. 100:257–268, 1990), and E. orontii (Plotnikova et al., Mycologia 90:1009–1016, 1998).
Both compatible and incompatible interactions between Erysiphe species and Arabidopsis ecotypes have been characterized (Adam et al., supra; Plotnikova et al., supra; Reuber et al., supra; and Xiao et al., The Plant Journal 12:757–768, 1997). A number of ecotypes exhibit R-avr gene-mediated resistance, although in some instances the resistance conferred by a single R gene is weak and the synergistic action of multiple R genes is required for an effective resistance response (Adam et al., supra and Xiao et al., supra).
On mature Arabidopsis leaves, asexual conidia of E. orontii germinate within 1–2 hours, appressoria begin to form by 5 hours, and by 24 hours development of haustoria is initiated (Plotnikova et al., supra). When infected by E. orontii, Arabidopsis expresses the pathogenesis related genes PR-1, PR-2 (BGL2), and PR-5 (Reuber et al., supra). It has previously been shown that the induction of these PR genes occurs at least partially via a SA (salicylic acid)-dependent pathway (reviewed in Yang et al., supra). The importance of this pathway in limiting E. orontii growth was demonstrated by an analysis of mutant lines that also exhibit increased susceptibility to the bacterial pathogen P. syringae (Reuber et al., supra). Mutants characterized by SA accumulation (pad4; Glazebrook et al., 1996; Jirage et al., Proc. Natl. Acad. Sci. U.S.A. 96:13583–13588, 1999; and Zhou et al., supra) and eds5; Nawrath et al., supra; Rogers et al., supra; and Volko et al., supra.) and an SA-deficient transgenic line expressing the bacterial nahG gene (Gaffney et al., supra) are more susceptible to E. orontii, as is the SA-unresponsive mutant npr1(Cao et al., supra, Delaney et al., supra; and Shah et al., supra). Furthermore, analysis of PR gene expression in mutant and transgenic lines suggested that all of the PR-1 mRNA accumulation that is elicited by E. orontii infection occurs via SA-dependent pathway(s), whereas both SA-dependent and SA-independent pathways contribute to BGL2 and PR-5 expression (Reuber et al., supra). An additional signal transduction pathway that is instrumental in defense against some pathogens requires the signaling molecules jasmonic acid (JA) and ethylene (ET) (Staswick et al., The Plant Journal 15:747–754, 1998 and Thomma et al., Plant Physiology 121:1093–1101, 1999 and reviewed in Chang et al., Current Opinion in Plant Biology 2:352–358, 1999; Creelman et al., Annual Review of Plant Physiology and Plant Molecular Biology 48:355–381, 1997; and Wastemack et al., Trends in Plant Science 2:302–307, 1997) and leads to the production of the antimicrobial proteins defensin and thionin (Epple et al., Plant Physiology 109:813–820, 1995 and Penninckx et al., 1996). In contrast to SA-inducible PR genes, the defensin gene PDF1.2 and the thionin gene THI2.1 are not expressed in E. orontii infected Arabidopsis (Reuber et al., supra).